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Sambrook PN, Geusens P, Ribot C, Solimano JA, Ferrer-Barriendos J, Gaines K, Verbruggen N, Melton ME (University of Sydney, Sydney, Australia; University Campus Limburg, Diepenbeek, Belgium; University Hospital, Maastricht, The Netherlands; CHU Toulouse Hôpital de Rangueil, Toulouse, France; Instituto Medico Miraflores, Lima, Peru; Hospital Universitario Central de Asturias, Oviedo, Spain; and Merck & Co., Inc., Whitehouse Station, NJ, USA). Alendronate produces greater effects than raloxifene on bone density and bone turnover in postmenopausal women with low bone density: results of EFFECT ( EFficacy of FOSAMAX® versus EVISTA® Comparison Trial) International. J Intern Med 2004; 255: 503–511. Alendronate and raloxifene are antiresorptive agents with different mechanisms of action, each used to treat osteoporosis in postmenopausal women. This study was undertaken to compare the efficacy and tolerability of alendronate to raloxifene in postmenopausal women with low-bone density. Randomized, double-masked, double-dummy multicentre international study. Clinical trial centres in Europe, South America and Asia-Pacific. A total of 487 postmenopausal women with low bone density, based on bone mineral density (BMD) of the lumbar spine or hip (T-score ≤−2.0). Patients received either alendronate 70 mg once weekly and daily placebo identical to raloxifene or raloxifene 60 mg daily and weekly placebo identical to alendronate for 12 months. Evaluations included BMD of the lumbar spine and hip and markers of bone turnover at 6 and 12 months and adverse event reporting. Alendronate demonstrated substantially greater increases in BMD than raloxifene at both lumbar spine and hip sites at 12 months. Lumbar spine BMD increased 4.8% with alendronate vs. 2.2% with raloxifene ( P < 0.001). The increase in total hip BMD was 2.3% with alendronate vs. 0.8% with raloxifene ( P < 0.001). Reductions in bone turnover were significantly larger with alendronate than raloxifene. Overall tolerability was similar, however, the proportion of patients reporting vasomotor events was significantly higher with raloxifene (9.5%) than with alendronate (3.7%, P = 0.010). The proportion of patients reporting gastrointestinal events was similar between groups. In postmenopausal women with low bone density, improvements in BMD and markers of bone turnover were substantially greater during treatment with alendronate compared to raloxifene. [ABSTRACT FROM AUTHOR]

Background: Natural populations of Arabidopsis thaliana exhibit phenotypic variations in specific environments and growth conditions. However, this variation has not been explored after seed osmopriming treatments. The natural variation in biomass production and root system architecture (RSA) was investigated across the Arabidopsis thaliana core collection in response to the pre-sawing seed treatments by osmopriming, with and without melatonin (Mel). The goal was to identify and characterize physiologically contrasting ecotypes., Results: Variability in RSA parameters in response to PEG-6000 seed osmopriming with and without Mel was observed across Arabidopsis thaliana ecotypes with especially positive impact of Mel addition under both control and 100 mM NaCl stress conditions. Two ecotypes, Can-0 and Kn-0, exhibited contrasted root phenotypes: seed osmopriming with and without Mel reduced the root growth of Can-0 plants while enhancing it in Kn-0 ones under both control and salt stress conditions. To understand the stress responses in these two ecotypes, main stress markers as well as physiological analyses were assessed in shoots and roots. Although the effect of Mel addition was evident in both ecotypes, its protective effect was more pronounced in Kn-0. Antioxidant enzymes were induced by osmopriming with Mel in both ecotypes, but Kn-0 was characterized by a higher responsiveness, especially in the activities of peroxidases in roots. Kn-0 plants experienced lower oxidative stress, and salt-induced ROS accumulation was reduced by osmopriming with Mel. In contrast, Can-0 exhibited lower enzyme activities but the accumulation of proline in its organs was particularly high. In both ecotypes, a greater response of antioxidant enzymes and proline accumulation was observed compared to mechanisms involving the reduction of Na + content and prevention of K + efflux., Conclusions: In contrast to Can-0, Kn-0 plants grown from seeds osmoprimed with and without Mel displayed a lower root sensitivity to NaCl-induced oxidative stress. The opposite root growth patterns, enhanced by osmopriming treatments might result from different protective mechanisms employed by these two ecotypes which in turn result from adaptive strategies proper to specific habitats from which Can-0 and Kn-0 originate. The isolation of contrasting phenotypes paves the way for the identification of genetic factors affecting osmopriming efficiency., (© 2024. The Author(s).)

Background: There is no standard definition of respiratory-related hospitalisation, a common end-point in idiopathic pulmonary fibrosis (IPF) clinical trials. As diverse aetiologies and complicating comorbidities can present similarly, external adjudication is sometimes employed to achieve standardisation of these events., Methods: An algorithm for respiratory-related hospitalisation was developed through a literature review of IPF clinical trials with respiratory-related hospitalisation as an end-point. Experts reviewed the algorithm until a consensus was reached. The algorithm was validated using data from the phase 3 ISABELA trials (clinicaltrials.gov identifiers NCT03711162 and NCT03733444), by assessing concordance between nonadjudicated, investigator-defined, respiratory-related hospitalisations and those defined by the adjudication committee using the algorithm., Results: The algorithm classifies respiratory-related hospitalisation according to cause: extraparenchymal (worsening respiratory symptoms due to left heart failure, volume overload, pulmonary embolism, pneumothorax or trauma); other (respiratory tract infection, right heart failure or exacerbation of COPD); "definite" acute exacerbation of IPF (AEIPF) (worsening respiratory symptoms within 1 month, with radiological or histological evidence of diffuse alveolar damage); or "suspected" AEIPF (as for "definite" AEIPF, but with no radiological or histological evidence of diffuse alveolar damage). Exacerbations ("definite" or "suspected") with identified triggers (infective, post-procedural or traumatic, drug toxicity- or aspiration-related) are classed as "known AEIPF"; "idiopathic AEIPF" refers to exacerbations with no identified trigger. In the ISABELA programme, there was 94% concordance between investigator- and adjudication committee-determined causes of respiratory-related hospitalisation., Conclusion: The algorithm could help to ensure consistency in the reporting of respiratory-related hospitalisation in IPF trials, optimising its utility as an end-point., Competing Interests: Conflict of interest: P. Ford and N. Prasad are former employees of, and have received warrants from, Galapagos. Conflict of interest: M. Kreuter reports grants and personal fees from Galapagos during the conduct of the study, and grants and personal fees from Boehringer Ingelheim and Roche outside the submitted work. Conflict of interest: K.K. Brown reports support from the National Heart, Lung, and Blood Institute; participation on a Scientific Advisory Board/work as an external science advisor for Blade Therapeutics, Boehringer Ingelheim, Bristol Myers Squibb, CSL Behring, DevPro Biopharma, Eleven P15, Galapagos, Galecto, Huitai Biomedicine, Open Source Imaging, Pliant, RedxPharma and Sanofi; participation on a data monitoring committee for Biogen and Humanetics; and work in a leadership or fiduciary role for the Fleischner Society and the Open Source Imaging Consortium. Conflict of interest: W.A. Wuyts reports no personal fees; the University Hospitals Leuven received consultancy fees from Galapagos during the conduct of the study, and grants from Boehringer Ingelheim and Roche outside the submitted work. Conflict of interest: M. Wijsenbeek reports no personal fees; the Erasmus MC received consultancy fees from AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, CSL Behring, Galapagos, Galecto, Hoffmann-La Roche, Horizon Therapeutics, Kinevant Sciences, Molecure, NeRRe Therapeutics, Novartis, PureTech Health, Thyron and Vicore; and grants from AstraZeneca/Daiichi Sankyo, Boehringer Ingelheim and Hoffmann-La Roche outside the submitted work. The Erasmus MC received speaker fees from Boehringer Ingelheim, CSL Behring and Hoffmann-La Roche outside the submitted work. Conflict of interest: D. Israël-Biet reports speaker and advisory board fees from Boehringer Ingelheim. Conflict of interest: R. Hubbard reports consulting fees from Galapagos. Conflict of interest: S.D. Nathan reports consulting fees from Bellerophon, Boehringer Ingelheim, Galapagos, Roche-Genentech and United Therapeutics. He is on the speakers’ bureau for Boehringer Ingelheim, Roche-Genentech and United Therapeutics. Conflict of interest: H. Nunes reports consulting fees from Galapagos during the conduct of the study, and consulting fees from Boehringer Ingelheim and Roche-Genentech. Conflict of interest: B. Penninckx is a former consultant to Galapagos. Conflict of interest: I. Seghers is a former employee of Galapagos. Conflict of interest: N. Verbruggen is an employee of Galapagos. Conflict of interest: P. Spagnolo reports consulting fees from Behring, Chiesi, Galapagos (during the conduct of this study), Glycocore, Lupin, Novartis, Pieris and PPM services, and institutional grants from Boehringer Ingelheim, PPM services and Roche outside the submitted work. Conflict of interest: N. Hirani reports no personal fees; the University of Edinburgh received fees from Boehringer Ingelheim, Galapagos, Guidepoint Global and Roche for consultancy and advisory work on behalf of Dr Hirani. The University of Edinburgh and NHS Lothian R&D have received grants and donations from Boehringer Ingelheim, Galecto, Indalo, Roche and UCB on behalf of Dr Hirani. No consultancies or grants are directly related to the submitted work. Conflict of interest: J. Behr reports personal fees from Galapagos during the conduct of the study, and consultation or advisory fees from AstraZeneca, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Novartis, Pliant, Roche and Sanofi/Genzyme; funding or grants from Boehringer Ingelheim; speaking and lecture fees from AstraZeneca, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Ferrer, Novartis, Pliant, Roche and Sanofi/Genzyme; and travel reimbursement from Boehringer Ingelheim, Ferrer and Roche outside the submitted work. Conflict of interest: R.J. Kaner reports personal fees from Galapagos during the conduct of the study, and grants from Afferent, Boehringer Ingelheim, Bristol Myers Squibb, Chiesi Pillar, CSL Behring, Genentech, National Institutes of Health, Pulmonary Fibrosis Foundation, Respivant and Toray, and personal fees from Boehringer Ingelheim, Genentech, Gilead, Medimmune, The France Foundation and United Therapeutics outside the submitted work. Conflict of interest: T.M. Maher, via his institution, has received industry-academic funding from AstraZeneca and GlaxoSmithKline R&D, and consultation or advisory fees from AstraZeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol Myers Squibb, CSL Behring, Fibrogen, Galapagos, Galecto, GlaxoSmithKline, IQVIA, Pliant, Roche, Trevi and Veracyte., (Copyright ©The authors 2024.)

The identification of transcription factor (TF) target genes is central in biology. A popular approach is based on the location by pattern matching of potential cis-regulatory elements (CREs). During the last few years, tools integrating next-generation sequencing data have been developed to improve the performance of pattern matching. However, such tools have not yet been comprehensively evaluated in plants. Hence, we developed a new streamlined method aiming at predicting CREs and target genes of plant TFs in specific organs or conditions. Our approach implements a supervised machine learning strategy, which allows decision rule models to be learnt using TF ChIP-chip/seq experimental data. Different layers of genomic features were integrated in predictive models: the position on the gene, the DNA sequence conservation, the chromatin state and various CRE footprints. Among the tested features, the chromatin features were crucial for improving the accuracy of the method. Furthermore, we evaluated the transferability of predictive models across TFs, organs and species. Finally, we validated our method by correctly inferring the target genes of key TFs controlling metabolite biosynthesis at the organ level in Arabidopsis. We developed a tool-Wimtrap-to reproduce our approach in plant species and conditions/organs for which ChIP-chip/seq data are available. Wimtrap is a user-friendly R package that supports an R Shiny web interface and is provided with pre-built models that can be used to quickly get predictions of CREs and TF gene targets in different organs or conditions in Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Zea mays., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Essential trace metals function as structural components or cofactors in many proteins involved in a wide range of physiological processes in plants. Hence, trace metal deficiency can significantly hamper plant growth and development. On the other hand, excess concentrations of trace metals can also induce phytotoxicity, for example via an enhanced production of reactive oxygen species. Besides their roles in plant growth under favourable environmental conditions, trace metals also contribute to plant responses to biotic and abiotic stresses. Heat is a stress factor that will become more prevalent due to increasing climate change and is known to negatively affect crop yield and quality, posing a severe threat to food security for future generations. Gaining insight into heat stress responses is essential to develop strategies to optimize plant growth and quality under unfavourable temperatures. In this context, trace metals deserve particular attention as they contribute to defence responses and are important determinants of plant nutritional value. Here, we provide an overview of heat-induced effects on plant trace metal homeostasis and the involvement of trace metals and trace metal-dependent enzymes in plant responses to heat stress. Furthermore, avenues for future research on the interactions between heat stress and trace metals are discussed., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Metallophytes constitute powerful models for the study of metal homeostasis, adaptation to extreme environments and the evolution of naturally selected traits. Arabidopsis halleri is a pseudometallophyte which shows constitutive zinc/cadmium (Zn/Cd) tolerance and Zn hyperaccumulation but high intraspecific variability in Cd accumulation. To examine the molecular basis of the variation in metal tolerance and accumulation, ionome, transcriptome and cell wall glycan array profiles were compared in two genetically close A. halleri populations from metalliferous and nonmetalliferous sites in Northern Italy. The metallicolous population displayed increased tolerance to and reduced hyperaccumulation of Zn, and limited accumulation of Cd, as well as altered metal homeostasis, compared to the nonmetallicolous population. This correlated well with the differential expression of transporter genes involved in trace metal entry and in Cd/Zn vacuolar sequestration in roots. Many cell wall-related genes were also more highly expressed in roots of the metallicolous population. Glycan array and histological staining analyses demonstrated that there were major differences between the two populations in terms of the accumulation of specific root pectin and hemicellulose epitopes. Our results support the idea that both specific cell wall components and regulation of transporter genes play a role in limiting accumulation of metals in A. halleri at contaminated sites., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The circadian clock coordinates the physiological responses of a biological system to day and night rhythms through complex loops of transcriptional/translational regulation. It can respond to external stimuli and adjust generated circadian oscillations accordingly to maintain an endogenous period close to 24 h. However, the interaction between nutritional status and circadian rhythms in plants is poorly understood. Magnesium (Mg) is essential for numerous biological processes in plants, and its homeostasis is crucial to maintain optimal development and growth. Magnesium deficiency in young Arabidopsis thaliana seedlings increased the period of circadian oscillations of the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter (pCCA1:LUC) activity and dampened their amplitude under constant light in a dose-dependent manner. Although the circadian period increase caused by Mg deficiency was light dependent, it did not depend on active photosynthesis. Mathematical modeling of the Mg input into the circadian clock reproduced the experimental increase of the circadian period and suggested that Mg is likely to affect global transcription/translation levels rather than a single component of the circadian oscillator. Upon addition of a low dose of cycloheximide to perturb translation, the circadian period increased further under Mg deficiency, which was rescued when sufficient Mg was supplied, supporting the model's prediction. These findings suggest that sufficient Mg supply is required to support proper timekeeping in plants., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

It has been proven challenging to conduct traditional efficacy trials for Ebola virus (EBOV) vaccines. In the absence of efficacy data, immunobridging is an approach to infer the likelihood of a vaccine protective effect, by translating vaccine immunogenicity in humans to a protective effect, using the relationship between vaccine immunogenicity and the desired outcome in a suitable animal model. We here propose to infer the protective effect of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen with an 8-week interval in humans by immunobridging. Immunogenicity and protective efficacy data were obtained for Ad26.ZEBOV and MVA-BN-Filo vaccine regimens using a fully lethal EBOV Kikwit challenge model in cynomolgus monkeys (nonhuman primates [NHP]). The association between EBOV neutralizing antibodies, glycoprotein (GP)-binding antibodies, and GP-reactive T cells and survival in NHP was assessed by logistic regression analysis. Binding antibodies against the EBOV surface GP were identified as the immune parameter with the strongest correlation to survival post EBOV challenge, and used to infer the predicted protective effect of the vaccine in humans using published data from phase I studies. The human vaccine-elicited EBOV GP-binding antibody levels are in a range associated with significant protection against mortality in NHP. Based on this immunobridging analysis, the EBOV GP-specific-binding antibody levels elicited by the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen in humans will likely provide protection against EBOV disease.

Hyperaccumulation and hypertolerance of Trace Metal Elements (TME) like Cd and Zn are highly variable in pseudo-metallophytes species. In this study we compared the impact of high Cd or Zn concentration on the photosynthetic apparatus of the Arabidopsis arenosa and Arabidopsis halleri pseudo-metallophytes growing on the same contaminated site in Piekary Slaskie in southern Poland. Plants were grown in hydroponic culture for 6 weeks, and then treated with 1.0 mM Cd or 5.0 mM Zn for 5 days. Chlorophyll a fluorescence and pigment content were measured after 0, 1, 2, 3, 4, and 5 days in plants grown in control and exposed to Cd or Zn treatments. Moreover, the effect of TME excess on the level of oxidative stress and gas-exchange parameters were investigated. In both plant species, exposure to high Cd or Zn induced a decrease in chlorophyll and an increase in anthocyanin contents in leaves compared to the control condition. After 5 days Cd treatment, energy absorbance, trapped energy flux and the percentage of active reaction centers decreased in both species. However, the dissipated energy flux in the leaves of A. arenosa was smaller than in A. halleri . Zn treatment had more toxic effect than Cd on electron transport in A. halleri compared with A. arenosa . A. arenosa plants treated with Zn excess did not react as strongly as in the Cd treatment and a decrease only in electron transport flux and percentage of active reaction centers compared with control was observed. The two species showed contrasting Cd and Zn accumulation. Cd concentration was almost 3-fold higher in A. arenosa leaves than in A. halleri . On the opposite, A. halleri leaves contained 3-fold higher Zn concentration than A. arenosa . In short, our results showed that the two Arabidopsis metallicolous populations are resistant to high Cd or Zn concentration, however, the photosynthetic apparatus responded differently to the toxic effects.

Introduction: While current standard of care (SOC) for idiopathic pulmonary fibrosis (IPF) slows disease progression, prognosis remains poor. Therefore, an unmet need exists for novel, well-tolerated agents that reduce lung function decline and improve quality of life. Here we report the design of two phase III studies of the novel IPF therapy, GLPG1690., Methods and Analysis: Two identically designed, phase III, international, randomised, double-blind, placebo-controlled, parallel-group, multicentre studies (ISABELA 1 and 2) were initiated in November 2018. It is planned that, in each study, 750 subjects with IPF will be randomised 1:1:1 to receive oral GLPG1690 600 mg, GLPG1690 200 mg or placebo, once daily, on top of local SOC, for at least 52 weeks. The primary endpoint is rate of decline of forced vital capacity (FVC) over 52 weeks. Key secondary endpoints are week 52 composite endpoint of disease progression or all-cause mortality (defined as composite endpoint of first occurrence of ≥10% absolute decline in per cent predicted FVC or all-cause mortality at week 52); time to first respiratory-related hospitalisation until end of study; and week 52 change from baseline in the St George's Respiratory Questionnaire total score (a quality-of-life measure)., Ethics and Dissemination: Studies will be conducted in accordance with Good Clinical Practice guidelines, Declaration of Helsinki principles, and local ethical and legal requirements. Results will be reported in a peer-reviewed publication., Trial Registration Numbers: NCT03711162; NCT03733444., Competing Interests: Competing interests: TMM has received personal fees from AstraZeneca, Bayer, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Galapagos, GlaxoSmithKline, Indalo Therapeutics, Pliant Therapeutics, ProMetic, Roche, Samumed and UCB, and stock options from Apellis outside the submitted work. His institution received grants or research fees from AstraZeneca, GlaxoSmithKline and UCB outside the submitted work. MK reports grants and personal fees from Galapagos during the conduct of the study, and grants and personal fees from Boehringer Ingelheim and Roche outside the submitted work. DJL reports personal fees from Galapagos during the conduct of the study, and personal fees from Philips Respironics, Roche, Sanofi Genzyme and Veracyte, grants and personal fees from Boehringer Ingelheim, FibroGen and Global Blood Therapeutics, unpaid Steering Committee membership for Galecto, and institutional fees for consultancy work from the Pulmonary Fibrosis Foundation outside the submitted work. KKB reports personal fees from Galapagos during the conduct of the study, and conversation under CDA with Genoa, grants from National Heart, Lung, and Blood Institute, and personal fees from Aeolus, AstraZeneca, aTyr, Biogen, Boehringer Ingelheim, Galapagos, Galecto Biotech, MedImmune, Novartis, Roche/Genentech, ProMetic, Patara and Third Pole outside the submitted work. WW reports no personal fees; the University Hospitals Leuven received consultancy fees from Galapagos during the conduct of the study and grants from Boehringer Ingelheim and Roche outside the submitted work. NV, SS, PF and WA-S are employees of, and have received warrants from, Galapagos. AF is an employee of Galapagos. MW reports no personal fees; the Erasmus MC received an advisory board fee from Galapagos, and speaker and advisory board fees from Boehringer Ingelheim and Hoffmann-La Roche outside the submitted work.

Ca 2+ signals in plant cells are important for adaptive responses to environmental stresses. Here, we report that the Arabidopsis CATION/Ca 2+ EXCHANGER2 (CCX2), encoding a putative cation/Ca 2+ exchanger that localizes to the endoplasmic reticulum (ER), is strongly induced by salt and osmotic stresses. Compared with the WT, AtCCX2 loss-of-function mutant was less tolerant to osmotic stress and displayed the most noteworthy phenotypes (less root/shoot growth) during salt stress. Conversely, AtCCX2 gain-of-function mutants were more tolerant to osmotic stress. In addition, AtCCX2 partially suppresses the Ca 2+ sensitivity of K667 yeast triple mutant, characterized by Ca 2+ uptake deficiency. Remarkably, Cameleon Ca 2+ sensors revealed that the absence of AtCCX2 activity results in decreased cytosolic and increased ER Ca 2+ concentrations in comparison with both WT and the gain-of-function mutants. This was observed in both salt and nonsalt osmotic stress conditions. It appears that AtCCX2 is directly involved in the control of Ca 2+ fluxes between the ER and the cytosol, which plays a key role in the ability of plants to cope with osmotic stresses. To our knowledge, Atccx2 is unique as a plant mutant to show a measured alteration in ER Ca 2+ concentrations. In this study, we identified the ER-localized AtCCX2 as a pivotal player in the regulation of ER Ca 2+ dynamics that heavily influence plant growth upon salt and osmotic stress., Competing Interests: The authors declare no conflict of interest.

Zinc (Zn) hyperaccumulation and hypertolerance are highly variable traits in Arabidopsis halleri. Metallicolous populations have evolved from nearby nonmetallicolous populations in multiple independent adaptation events. To determine whether these events resulted in similar or divergent adaptive strategies to high soil Zn concentrations, we compared two A. halleri metallicolous populations from distant genetic units in Europe (Poland (PL22) and Italy (I16)). The ionomic (Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)) and transcriptomic (RNA sequencing (RNA-Seq)) responses to growth at 5 and 150 μM Zn were analyzed in root and shoot tissues to examine the contribution of the geographic origin and treatment to variation among populations. These analyses were enabled by the generation of a reference A. halleri transcriptome assembly. The genetic unit accounted for the largest variation in the gene expression profile, whereas the two populations had contrasting Zn accumulation phenotypes and shared little common response to the Zn treatment. The PL22 population displayed an iron deficiency response at high Zn in roots and shoots, which may account for higher Zn accumulation. By contrast, I16, originating from a highly Zn-contaminated soil, strongly responded to control conditions. Our data suggest that distinct mechanisms support adaptation to high Zn in soils among A. halleri metallicolous populations., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

While cadmium (Cd) tolerance is a constitutive trait in the Arabidopsis halleri species, Cd accumulation is highly variable. Recent adaptation to anthropogenic metal stress has occurred independently within the genetic units of A. halleri and the evolution of different mechanisms involved in Cd tolerance and accumulation has been suggested. To gain a better understanding of the mechanisms underlying Cd tolerance and accumulation in A. halleri, ionomic inductively coupled plasma mass spectrometry (ICP-MS), transcriptomic (RNA sequencing) and metabolomic (high-performance liquid chromatography-mass spectrometry) profiles were analysed in two A. halleri metallicolous populations from different genetic units (PL22 from Poland and I16 from Italy). The PL22 and I16 populations were both hypertolerant to Cd, but PL22 hyperaccumulated Cd while I16 behaved as an excluder both in situ and when grown hydroponically. The observed hyperaccumulator vs excluder behaviours were paralleled by large differences in the expression profiles of transporter genes. Flavonoid-related transcripts and metabolites were strikingly more abundant in PL22 than in I16 shoots. The role of novel A. halleri candidate genes possibly involved in Cd hyperaccumulation or exclusion was supported by the study of corresponding A. thaliana knockout mutants. Taken together, our results are suggestive of the evolution of divergent strategies for Cd uptake, transport and detoxification in different genetic units of A. halleri., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

This review synthesizes contemporary understanding of copper-cobalt (Cu-Co) tolerance and accumulation in plants. Accumulation of foliar Cu and Co to > 300 μg g -1 is exceptionally rare globally, and known principally from the Copperbelt of Central Africa. Cobalt accumulation is also observed in a limited number of nickel (Ni) hyperaccumulator plants occurring on ultramafic soils around the world. None of the putative Cu or Co hyperaccumulator plants appears to comply with the fundamental principle of hyperaccumulation, as foliar Cu-Co accumulation is strongly dose-dependent. Abnormally high plant tissue Cu concentrations occur only when plants are exposed to high soil Cu with a low root to shoot translocation factor. Most Cu-tolerant plants are Excluders sensu Baker and therefore setting nominal threshold values for Cu hyperaccumulation is not informative. Abnormal accumulation of Co occurs under similar circumstances in the Copperbelt of Central Africa as well as sporadically in Ni hyperaccumulator plants on ultramafic soils; however, Co-tolerant plants behave physiologically as Indicators sensu Baker. Practical application of Cu-Co accumulator plants in phytomining is limited due to their dose-dependent accumulation characteristics, although for Co field trials may be warranted on highly Co-contaminated mineral wastes because of its relatively high metal value., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

As a drastic environmental change, metal pollution may promote the rapid evolution of genetic adaptations contributing to metal tolerance. In Arabidopsis halleri, genetic bases of zinc (Zn) and cadmium (Cd) tolerance have been uncovered only in a metallicolous accession, although tolerance is species-wide. The genetic determinants of Zn and Cd tolerance in a nonmetallicolous accession were thus investigated for the first time. The genetic architecture of tolerance was investigated in a nonmetallicolous population (SK2) by using first backcross progeny obtained from crosses between SK2 and Arabidopsis lyrata petraea, a nonmetallophyte species. Only one significant and common quantitative trait locus (QTL) region was identified explaining 22.6% and 31.2% of the phenotypic variation for Zn and Cd tolerance, respectively. This QTL co-localized with HEAVY METAL ATPASE 4 (AhHMA4), which was previously validated as a determinant of Zn and Cd tolerance in a metallicolous accession. Triplication and high expression of HMA4 were confirmed in SK2. In contrast, gene duplication and high expression of METAL TOLERANT PROTEIN 1A (MTP1A), which was previously associated with Zn tolerance in a metallicolous accession, were not observed in SK2. Overall, the results support the role of HMA4 in tolerance capacities of A. halleri that may have pre-existed in nonmetallicolous populations before colonization of metal-polluted habitats. Preadaptation to metal-contaminated sites is thus discussed., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Investigation of genetic determinants of Cd tolerance in the Zn/Cd hyperaccumulator Arabidopsis halleri allowed the identification of the vacuolar Ca(2+)/H(+) exchanger encoding CAX1 gene. CAX1 was proposed to interfere with the positive feedback loop between Reactive Oxygen Species (ROS) production and Cd-induced cytosolic Ca(2+) spikes, especially at low external Ca(2+) supply. In this study expression of genes involved in ROS homeostasis, cell wall composition, apoplastic pH regulation and Ca(2+) homeostasis were monitored in Arabidopsis thaliana wild-type and cax1-1 knock-out mutant and in Arabidopsis halleri wild-type exposed to cadmium or in control conditions. Clustering the outputs of the expression analysis in a gene co-expression network revealed that CAX1 and genes involved in Ca(2+) cellular homeostasis, apoplastic pH and oxidative stress response were highly correlated in A. thaliana, but not in A. halleri. Many of the studied genes were already highly expressed in A. halleri and/or their expression was not modified by exposure to Cd. The results further supported the role of CAX1 in the regulation of cytosolic ROS accumulation as well as the existence of different cell wall modifications strategies in response to Cd in Arabidopsis thaliana and halleri.

An original approach to develop sustainable agriculture with less nitrogen fertilizer inputs is to tackle the cross-talk between nitrogen nutrition and plant growth regulators. In particular the gaseous hormone, ethylene, is a prime target for that purpose. The variation of ethylene production in natural accessions of the model species Arabidopsis thaliana was explored in response to the nitrate supply. Ethylene was measured with a laser-based photoacoustic detector. First, experimental conditions were established with Columbia-0 (Col-0) accession, which was grown in vitro on horizontal plates across a range of five nitrate concentrations (0.5, 1, 2.5, 5, or 10 mM). The concentrations of 1 and 10 mM nitrate were retained for further characterization. Along with a decrease of total dry biomass and higher biomass allocation to the roots, the ethylene production was 50% more important at 1 mM than at 10 mM nitrate. The total transcript levels of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASES (ACS) in roots and those of ACC OXIDASES (ACO) in shoots increased by 100% between the same treatments. This was mainly due to higher transcript levels of ACS6 and of ACO2 and ACO4 respectively. The assumption was that during nitrogen deficiency, the greater biomass allocation in favor of the roots was controlled by ethylene being released in the shoots after conversion of ACC originating from the roots. Second, biomass and ethylene productions were measured in 20 additional accessions. Across all accessions, the total dry biomass and ethylene production were correlated negatively at 1 mM but positively at 10 mM nitrate. Furthermore, polymorphism was surveyed in ACC and ethylene biosynthesis genes and gene products among accessions. Very few substitutions modifying the amino acids properties in conserved motifs of the enzymes were found in the accessions. Natural variation of ethylene production could be further explored to improve Nitrogen Use Efficiency (NUE), in particular by manipulating features like the biomass production and the timing of senescence upon nitrogen limitation.

The circadian clock is an endogenous timekeeper that allows organisms to anticipate and adapt to the daily variations of their environment. The plant clock is an intricate network of interlocked feedback loops, in which transcription factors regulate each other to generate oscillations with expression peaks at specific times of the day. Over the last decade, mathematical modeling approaches have been used to understand the inner workings of the clock in the model plant Arabidopsis thaliana. Those efforts have produced a number of models of ever increasing complexity. Here, we present an alternative model that combines a low number of equations and parameters, similar to the very earliest models, with the complex network structure found in more recent ones. This simple model describes the temporal evolution of the abundance of eight clock gene mRNA/protein and captures key features of the clock on a qualitative level, namely the entrained and free-running behaviors of the wild type clock, as well as the defects found in knockout mutants (such as altered free-running periods, lack of entrainment, or changes in the expression of other clock genes). Additionally, our model produces complex responses to various light cues, such as extreme photoperiods and non-24 h environmental cycles, and can describe the control of hypocotyl growth by the clock. Our model constitutes a useful tool to probe dynamical properties of the core clock as well as clock-dependent processes.

The mitotic checkpoint (MC) guards faithful sister chromatid segregation by monitoring the attachment of spindle microtubules to the kinetochores. When chromosome attachment errors are detected, MC delays the metaphase-to-anaphase transition through the inhibition of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. In contrast to yeast and mammals, our knowledge on the proteins involved in MC in plants is scarce. Transient synchronization of root tips as well as promoter-reporter gene fusions were performed to analyze temporal and spatial expression of COPPER MODIFIED RESISTANCE1/PATRONUS1 (CMR1/PANS1) in developing Arabidopsis thaliana seedlings. Functional analysis of the gene was carried out, including CYCB1;2 stability in CMR1/PANS1 knockout and overexpressor background as well as metaphase-anaphase chromosome status. CMR1/PANS1 is transcriptionally active during M phase. Its deficiency provokes premature cell cycle exit and in consequence a rapid consumption of the number of meristematic cells in particular under stress conditions that are known to affect spindle microtubules. Root growth impairment is correlated with a failure to delay the onset of anaphase, resulting in anaphase bridges and chromosome missegregation. CMR1/PANS1 overexpression stabilizes the mitotic CYCB1;2 protein. Likely, CMR1/PANS1 coordinates mitotic cell cycle progression by acting as an APC/C inhibitor and plays a key role in growth adaptation to stress., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Certain molecular mechanisms of Cd tolerance and accumulation have been identified in the model species Arabidopsis halleri, while intraspecific variability of these traits and the mechanisms of shoot detoxification were little addressed. The Cd tolerance and accumulation of metallicolous and non-metallicolous A. halleri populations from different genetic units were tested in controlled conditions. In addition, changes in shoot cell wall composition were investigated using Fourier transform infrared spectroscopy. Indeed, recent works on A. halleri suggest Cd sequestration both inside cells and in the cell wall/apoplast. All A. halleri populations tested were hypertolerant to Cd, and the metallicolous populations were on average the most tolerant. Accumulation was highly variable between and within populations, and populations that were non-accumulators of Cd were identified. The effect of Cd on the cell wall composition was quite similar in the sensitive species A. lyrata and in A. halleri individuals; the pectin/polysaccharide content of cell walls seems to increase after Cd treatment. Nevertheless, the changes induced by Cd were more pronounced in the less tolerant individuals, leading to a correlation between the level of tolerance and the extent of modifications. This work demonstrated that Cd tolerance and accumulation are highly variable traits in A. halleri, suggesting adaptation at the local scale and involvement of various molecular mechanisms. While in non-metallicolous populations drastic modifications of the cell wall occur due to higher Cd toxicity and/or Cd immobilization in this compartment, the increased tolerance of metallicolous populations probably involves other mechanisms such as vacuolar sequestration., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Arabidopsis halleri is a model plant for Zn and Cd hyperaccumulation. The objective of this study was to determine the relationship between the chemical forms of Cd, its distribution in leaves, and Cd accumulation and tolerance. An interspecific cross was carried out between A. halleri and the non-tolerant and non-hyperaccumulating relative A. lyrata providing progenies segregating for Cd tolerance and accumulation. Cd speciation and distribution were investigated using X-ray absorption spectroscopy and microfocused X-ray fluorescence. In A. lyrata and non-tolerant progenies, Cd was coordinated by S atoms only or with a small contribution of O groups. Interestingly, the proportion of O ligands increased in A. halleri and tolerant progenies, and they were predominant in most of them, while S ligands were still present. Therefore, the binding of Cd with O ligands was associated with Cd tolerance. In A. halleri, Cd was mainly located in the xylem, phloem, and mesophyll tissue, suggesting a reallocation process for Cd within the plant. The distribution of the metal at the cell level was further discussed. In A. lyrata, the vascular bundles were also Cd enriched, but the epidermis was richer in Cd as compared with the mesophyll. Cd was identified in trichomes of both species. This work demonstrated that both Cd speciation and localization were related to the tolerance character of the plant., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The cathepsin K inhibitor odanacatib (ODN), currently in phase 3 development for postmenopausal osteoporosis, has a novel mechanism of action that reduces bone resorption while maintaining bone formation. In phase 2 studies, odanacatib increased areal bone mineral density (aBMD) at the lumbar spine and total hip progressively over 5 years. To determine the effects of ODN on cortical and trabecular bone and estimate changes in bone strength, we conducted a randomized, double-blind, placebo-controlled trial, using both quantitative computed tomography (QCT) and high-resolution peripheral (HR-p)QCT. In previously published results, odanacatib was superior to placebo with respect to increases in trabecular volumetric BMD (vBMD) and estimated compressive strength at the spine, and integral and trabecular vBMD and estimated strength at the hip. Here, we report the results of HR-pQCT assessment. A total of 214 postmenopausal women (mean age 64.0 ± 6.8 years and baseline lumbar spine T-score -1.81 ± 0.83) were randomized to oral ODN 50 mg or placebo, weekly for 2 years. With ODN, significant increases from baseline in total vBMD occurred at the distal radius and tibia. Treatment differences from placebo were also significant (3.84% and 2.63% for radius and tibia, respectively). At both sites, significant differences from placebo were also found in trabecular vBMD, cortical vBMD, cortical thickness, cortical area, and strength (failure load) estimated using finite element analysis of HR-pQCT scans (treatment differences at radius and tibia = 2.64% and 2.66%). At the distal radius, odanacatib significantly improved trabecular thickness and bone volume/total volume (BV/TV) versus placebo. At a more proximal radial site, odanacatib attenuated the increase in cortical porosity found with placebo (treatment difference = -7.7%, p = 0.066). At the distal tibia, odanacatib significantly improved trabecular number, separation, and BV/TV versus placebo. Safety and tolerability were similar between treatment groups. In conclusion, odanacatib increased cortical and trabecular density, cortical thickness, aspects of trabecular microarchitecture, and estimated strength at the distal radius and distal tibia compared with placebo., (© 2014 American Society for Bone and Mineral Research.)

The exposure of plants to high concentrations of trace metallic elements such as copper involves a remodeling of the root system, characterized by a primary root growth inhibition and an increase in the lateral root density. These characteristics constitute easy and suitable markers for screening mutants altered in their response to copper excess. A forward genetic approach was undertaken in order to discover novel genetic factors involved in the response to copper excess. A Cu(2+) -sensitive mutant named copper modified resistance1 (cmr1) was isolated and a causative mutation in the CMR1 gene was identified by using positional cloning and next-generation sequencing. CMR1 encodes a plant-specific protein of unknown function. The analysis of the cmr1 mutant indicates that the CMR1 protein is required for optimal growth under normal conditions and has an essential role in the stress response. Impairment of the CMR1 activity alters root growth through aberrant activity of the root meristem, and modifies potassium concentration and hormonal balance (ethylene production and auxin accumulation). Our data support a putative role for CMR1 in cell division regulation and meristem maintenance. Research on the role of CMR1 will contribute to the understanding of the plasticity of plants in response to changing environments., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The mechanistic understanding of metal hyperaccumulation has benefitted immensely from the use of molecular genetics tools developed for Arabidopsis thaliana. The revolution in DNA sequencing will enable even greater strides in the near future, this time not restricted to the family Brassicaceae. Reference genomes are within reach for many ecologically interesting species including heterozygous outbreeders. They will allow deep RNA-seq transcriptome studies and the re-sequencing of contrasting individuals to unravel the genetic basis of phenotypic variation. Cell-type specific transcriptome analyses, which will be essential for the dissection of metal translocation pathways in hyperaccumulators, can be achieved through the combination of RNA-seq and translatome approaches. Affordable high-resolution genotyping of many individuals enables the elucidation of quantitative trait loci in intra- and interspecific crosses as well as through genome-wide association mapping across large panels of accessions. Furthermore, genome-wide scans have the power to detect loci under recent selection. Together these approaches will lead to a detailed understanding of the evolutionary path towards the emergence of hyperaccumulation traits.